Mitogen activated protein kinase phosphatase 5 alleviates liver ischemia–reperfusion injury by inhibiting TAK1/JNK/p38 pathway

Mitogen activated protein kinase phosphatase 5 (MKP5) is a member of the MKP family and has been implicated in diverse biological and pathological conditions. However, it is unknown what role MKP5 plays in liver ischemia/reperfusion (I/R) injury. In the present study, we used MKP5 global knockout (KO) and MKP5 overexpressing mice to establish a liver I/R injury model in vivo, and MKP5 knockdown or MKP5 overexpressing HepG2 cells to establish a hypoxia-reoxygenation (H/R) model in vitro. In this study we demonstrated that protein expression of MKP5 was significantly downregulated in liver tissue of mice after I/R injury, and HepG2 cells subjected to H/R injury. MKP5 KO or knockdown significantly increased liver injury, as demonstrated by elevated serum transaminases, hepatocyte necrosis, infiltrating inflammatory cells, secretion of pro-inflammatory cytokines, apoptosis, oxidative stress. Conversely, MKP5 overexpression significantly attenuated liver and cell injury. Furthermore, we showed that MKP5 exerted its protective effect by inhibiting c-Jun N-terminal kinase (JNK)/p38 activity, and its action was dependent on Transforming growth factor-β-activated kinase 1 (TAK1) activity. According to our results, MKP5 inhibited the TAK1/JNK/p38 pathway to protect liver from I/R injury. Our study identifies a novel target for the diagnosis and treatment of liver I/R injury.

Liver I/R model. We established a 70% I/R model in the mouse liver as previously described 27 . Briefly, after the mice were anesthetized with sodium pentobarbital, the abdominal cavity was opened, and the blood vessels of the left and middle hepatic lobes were separated and clamped with vascular clips. After 1.5 h of ischemia, the vascular clips were removed to start reperfusion. Mice in the sham-operated group received the same procedure without vessel clipping. Blood and liver tissue samples were collected after different time points of reperfusion.
Liver damage assessment. After reperfusion, the blood samples of mice were collected, and the supernatant was collected after centrifugation at 3500 × g for 5 min, and the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) (JianChen Bioengineering Institute, Nanjing, China) were measured according to the instructions of the detection kit.
Hematoxylin and eosin staining. Liver tissues were fixed with 10% formalin, embedded in paraffin, and cut into 5-μm thick paraffin sections, and stained with hematoxylin and eosin according to the kit instructions (Servicebio, Wuhan, China). The stained sections were photographed and analyzed under an inverted optical microscope (Olympus, Tokyo, Japan).
Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Paraffinembedded liver tissues were cut into 5-μm thick paraffin sections, and apoptosis was detected according to the instructions of the TUNEL kit (Servicebio, Wuhan, China). After TUNEL staining, the slices were sealed with anti-fluorescent quenching agent, and then observed and photographed with a fluorescence microscope (Olympus, Tokyo, Japan). The number of TUNEL positive (the nuclei are stained red) cells were recorded from five non-overlapping fields in each slice, and the apoptosis rate was calculated.
Immunohistochemical staining. Paraffin-embedded liver tissue sections were treated with xylene dewaxing and gradient ethanol dehydration, followed by repair solution, 3% H 2 O 2 inactivation of endogenous peroxidase, and sections were sealed with 10% goat serum. Tissue sections were incubated with Ly6g (1:200, Servicebio, Wuhan, China) and F4/80 (1:200, Servicebio, Wuhan, China) primary antibodies overnight at 4 °C. After washing with PBS for three times, the sections were incubated with goat anti-rabit secondary antibody at room temperature for 30 min, followed by dropwise addition of DAB chromogenic solution. The sections were re-stained with hematoxylin, sealed with neutral balsam, and observed and photographed under the microscope.

Quantitative real-time polymerase chain reaction (RT-PCR). Total RNA was extracted with
TriQuick reagent (Solarbio, Beijing, China), and cDNA was synthesized according to the instructions of reverse transcription kit (Vazyme, Nanjing, China). The reaction system was prepared for amplification according to the instructions of the RT-PCR kit (Vazyme, Nanjing, China). Amplification conditions were as follows: Pre-denaturation at 95˚C for 30 s, denaturation at 95˚C for 5 s, annealing and extension at 60˚C for 30 s, for a total of 40 cycles. The results were analyzed by the 2 −△△Ct method, and the expression of mRNA was expressed as a ratio to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primer sequences are shown in Supplementary Table 1.
Western blotting. The total protein in liver tissues and cells were extracted with RIPA reagent (Solarbio, Beijing, China), and the protein concentration was detected by BCA kit (Solarbio, Beijing, China), and then separated by 10% or 12% sodium dodecyl sulfonate-polyacrylamide gel electrophoresis. After electrophoresis, the gel was transferred to a PVDF membrane and blocked in 5% skimmed milk powder for 1 h at room temperature. Membranes were incubated with primary antibodies at 4 °C overnight. Membranes were washed with TBST and incubated with goat anti-rabbit or anti-mouse secondary antibody at 37 °C for 1 h. The ECL chemiluminescence reagent (NCM Biotech, Suzhou, China) was used for development, and the gel imaging system (Bio-Rad, CA, www.nature.com/scientificreports/ USA) was used for exposure and photography. The information for all antibodies used are described in Supplementary Table 2.
Oxidative stress analysis. For DHE Staining, after reperfusion, liver tissue samples were taken and embedded with optimal cutting temperature compound, frozen and fixed quickly. Tissue specimens were cut into 10 μm sections, and then stained with 10 μM dihydroethidium (DHE) fluorescent probe and incubated at room temperature for 1 h in the dark. After staining nuclei with DAPI, the sections were observed and photographed with a fluorescence microscope. For MDA, SOD and GSH detection, after weighing the mouse liver tissue, add 0.9% normal saline pre-cooled at 4 °C according to the ratio of mass (g) : volume (mL) to 1:9 to prepare 10% liver homogenate, centrifuged at 8000 × g for 10 min to collect the supernatant and use the BCA protein concentration assay kit to quantify the protein concentration, and operate accordance with the kit instructions to detect the levels of MDA, SOD and GSH (Solarbio, Beijing, China) in the liver tissue of each group.
Cell culture and cell H/R model. HepG2   and expressed as mean ± standard deviation. T-test was used for comparison between two groups, and one-way ANOVA analysis was used for comparison between more than two groups. P < 0.05 was considered statistically significant.

MKP5 is involved in the regulation of liver I/R injury in mice.
To analyze the role of MKP5 in liver I/R injury, we evaluated the protein expression of MKP5 in liver tissues after I/R injury and in HepG2 cells challenged by H/R injury. We found that the protein expression of MKP5 was significantly downregulated in vivo and in vitro ( Fig. 1A-D). The expression of MKP5 was the lowest at 6 h of reperfusion and 6 h of reoxygenation, and these two time points were selected for subsequent experiments. Next, we used MKP5 KO mice and AAV-MKP5 mice to investigate whether MKP5 regulates liver IR injury. Western blot analysis confirmed MKP5 KO or overexpression in the liver ( Fig. 1E and F). Notably, compared to wildtype (WT) mice, liver IR-injured mice with MKP5 KO displayed significantly higher serum levels of ALT, AST and LDH ( Fig. 1G-I). Additionally, liver sections from MKP5 KO mice showed more severe necrosis after liver I/R injury compared to WT mice ( Fig. 1J and K). Furthermore, compared to AAV-GFP group mice, serum levels of AST, ALT and LDH (Fig. 1L-N), and liver tissue necrosis were reduced in AAV-MKP5 group mice ( Fig. 1O and P). These results suggest that MKP5 protects the liver from injury caused by I/R. www.nature.com/scientificreports/ www.nature.com/scientificreports/ MKP5 reduces inflammatory response after liver I/R injury. During liver I/R injury, the inflammatory response plays a pivotal role in the overall I/R process, and inhibition of inflammation can effectively alleviate liver I/R injury. Therefore, we tested the role of MKP5 in the inflammatory response in our models. Compared to WT mice, mRNA expression of interleukin 6 (Il-6), interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), and monocyte chemoattractant protein-1 (MCP-1) were increased in liver tissues of MKP5 KO mice after I/R injury ( Fig. 2A-D). In addition, after liver I/R injury, immunohistochemistry staining showed a significant increase in the number of infiltrating inflammatory cells (Ly6g + cells and F4/80 + ) in MKP5 KO mice compared to WT mice ( Fig. 2E and F). We further demonstrated that MKP5 KO enhanced the activation of the nuclear factor kappa B (NF-κB) signaling pathway, shown by elevated levels of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα) and p65 phosphorylation, compared with WT mice (Fig. 2G and H). In contrast, AAV-MKP5 group mice inhibited the expression of inflammatory factors, inflammatory cell infiltration, and NF-κB signaling pathway compared with AAV-GFP group mice ( Fig. 2I-P). Collectively, these suggest that MKP5 is an important regulator of the inflammatory response during liver I/R injury.
MKP5 attenuates apoptosis after liver I/R injury. Apoptosis during liver I/R injury is directly involved in liver injury. TUNEL staining of liver tissue after I/R injury showed a significant increase in the number of apoptotic cells in MKP5 KO mice compared with WT mice (Fig. 3A and B). In addition, the expression of proapoptotic protein cleaved caspase-3 and B-cell lymphoma protein 2 (BCL-2)-associated X protein (BAX) were also significantly increased, while the expression of anti-apoptotic protein BCL-2 was significantly decreased in MKP5 KO mice after I/R injury ( Fig. 3C and D). In contrast, compared to AAV-GFP group mice, AAV-MKP5 group mice had decreased apoptosis after liver I/R injury, demonstrated by decreased TUNEL-positive cell numbers, reduced protein expression of BAX and cleaved caspase-3, and increased protein expression of BCL-2 ( Fig. 3E-H). Overall, these datas suggest that MKP5 inhibits hepatocyte apoptosis in liver I/R injury.
MKP5 reduces oxidative stress after liver I/R injury. SOD, GSH and MDA are important indexes of oxidative stress, DHE staining can be used to detect the content of ROS. Compared with the sham group, the activities of SOD and GSH in liver tissue were significantly decreased, and the content of MDA and ROS production were significantly increased after liver I/R (Fig. 4A-C). Compared with WT mice, MKP5 KO mice had significantly increased MDA content, ROS production, and decreased SOD and GSH activity in the liver after I/R injury (Fig. 4A-D). In contrast, MKP5 overexpression reduced significantly the MDA content, ROS production and increased SOD and GSH activity compared to AAV-GFP group mice ( Fig. 4E and H). These results demonstrate that MKP5 suppresses oxidative stress after liver I/R.

MKP5 attenuates cell damage after H/R injury. We further examined the function of MKP5 in
HepG2 cells subjected to H/R stimulation. Lentivirus containing either MKP5 shRNA or MKP5 expression plasmid were used to establish MKP5 knockdown or overexpression cells, respectively. Cell viability, LDH content in medium, cell apoptosis, and the expression of apoptosis-related proteins were detected. The knockdown and overexpression of MKP5 was verified by western blot (Fig. 5A-C), the lentivirus sh-2 sequence knockdown effect was the most obvious, which was used for subsequent experiments. Compared to NC group cells, MKP5 knockdown cells exposed to H/R resulted in significantly reduced cell viability, significantly increased LDH content and apoptosis (Fig. 5D-E and H-K). However, compared with flag group cells, H/R-treated HepG2 cells overexpressing MKP5 showed increased viability, LDH production, and decreased apoptosis (Fig. 5F-G and L-O). These results suggest that MKP5 has a protective effect against H/R-induced hepatocyte injury.

MKP5 inhibits the TAK1/JNK/p38 signaling pathways after liver I/R injury. The MAPK signaling
pathway is involved in regulating inflammation, apoptosis and oxidative stress after liver I/R injury. MKP5, a member of the MKP family, negatively regulates the activity of the MAPK pathway. Therefore, we speculated that MKP5 might protect liver from I/R injury by inhibiting the MAPK pathway. As shown in Fig. 6A and B, compared to WT mice, the protein levels of phosphorylated p38 and JNK were both upregulated in MKP5 KO mouse liver tissues after I/R injury. We then examined the expression of TAK1 an upstream protein of the MAPK signaling pathway. MKP5 KO mice had increased phosphorylated TAK1 expression in liver tissues after I/R injury compared to WT mice ( Fig. 6A and B). In contrast, compared to AAV-GFP group mice, AAV-MKP5 group mice had reduced phosphorylation of TAK1, and reduced expression of p38 and JNK ( Fig. 6C and D). Consistent with the in vivo results, we found the similar trend in HepG2 cells subjected to H/R injury (Fig. 6E-H). Together, these observations suggeste that in hepatocytes, MKP5 inhibits the activation of TAK1/JNK/p38 pathway during liver I/R-induced liver injury.

Effect of MKP5 on liver I/R depends on TAK1 activity.
To evaluated whether the protective effect of MKP5 was dependent on TAK1, we used a specific TAK1 inhibitor (5Z-7-ox) to block the activity of TAK1 and inhibit activation of downstream JNK/p38 signalling in MKP5 knockdown HepG2 cells prior to H/R injury. Compared with vehicle treated cells, 5Z-7-ox inhibited the phosphorylation of TAK1 and JNK/p38 in MKP5 knockdown HepG2 cells subjected to H/R challenge ( Fig. 7A and B). Furthermore, TAK1 inhibition also reversed the cell viability decrease, LDH release and apoptosis caused by MKP5 knockdown in HepG2 cells subjected to H/R (Fig. 7C-H). These observations suggest that TAK1 inhibition could eradicate the effects of MKP5 knockdown on H/R-induced cell injury, suggesting that TAK1 mediates the protective effect of MKP5 on liver I/R injury. www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/

Discussion
In the present study, we found that MKP5 expression was significantly reduced after liver I/R injury and in HepG2 cells H/R. MKP5 knockdown exacerbated liver injury via promoting inflammation, apoptosis, and oxidative stress. In contrast, overexpression of MKP5 significantly attenuated liver injury. Further analysis showed that MKP5 protected against liver I/R injury through regulating the TAK1/JNK/p38 pathway, and inhibition of TAK1 activity could reduce the cellular injury caused by MKP5 knockdown in HepG2 cells subjected to H/R. Inflammation and apoptosis play an important roles in liver I/R injury 16,17,29 . During liver I/R injury, activated Kupffer cells produce reactive oxygen species, pro-inflammatory cytokines, chemokines, and adhesion molecules, leading to cell apoptosis and tissue damage 8 . Consequently, the damaged hepatocytes promote more inflammatory cell infiltration, leading to a continuous vicious cycle that aggravates liver injury 13,15 . Targeting www.nature.com/scientificreports/ inflammatory response and apoptosis pathways during this process will significantly improve the prognosis of clinical I/R injury 29 . In our study, MKP5 KO led to a significant increase in the expression of pro-inflammatory factors (IL-1β, IL-6, TNF-α and MCP-1), and Ly6g and F4/80 positive cell infiltration. Furthermore, NF-κB pathway activation was more pronounced in liver tissues of MKP5 KO mice compared to the WT mice. TUNEL staining and apoptosis-related proteins BAX, BCL-2, and cleaved caspase-3 showed that MKP5 KO increased liver I/R-induced apoptosis. In contrast, compared with AAV-GFP group mice exposed to I/R, AAV-MKP5 group mice had reduced liver inflammation and apoptosis. Thus, we concluded that MKP5 could significantly reduce liver I/R-induced inflammation and apoptosis. A large number of oxygen radicals are generated in liver tissue during I/R injury 30 . Oxygen radicals and unsaturated fatty acids on biological membranes undergo lipid peroxidation to form lipid peroxides such as MDA, which is cytotoxic and can aggravate cell membrane damage and affect normal physiological functions of cells 30 . The important antioxidant enzymes SOD and GSH can scavenge oxygen free radicals and reduce the damage of lipid peroxidation in cells. Therefore, MDA content, SOD and GSH activity are often used to reflect the oxidative stress level of the body or tissues 31,32 . Dihydroethidium (DHE) can freely enter the cell and is oxidized by intracellular ROS to produce red fluorescence, which can be used to judge the amount and change of cellular ROS content 33,34 . Qian et al. showed that MKP5 attenuates LPS-induced vascular injury by inhibiting ROS production 35 . Zhao et al. demonstrated that MKP5 alleviates PA-induced islet β cell dysfunction by inhibiting oxidative stress 26 . These findings are consistent with the results shown in our study that MKP5 KO resulted in increased ROS production and MDA content, and decreasd SOD and GSH activity in liver tissues after I/R injury. While overexpression of MKP5 decreased the ROS production and MDA content, and increased SOD and GSH activity, suggesting that MKP5 inhibits oxidative stress after liver I/R injury.
The MAPK family consists of extracellular regulatory protein kinases (ERK), JNK, and p38, which are involved in the physiological processes of cell proliferation, apoptosis, inflammatory response, oxidative stress and other physiological processes in liver I/R injury 36,37 . Inhibition of MAPK activity is a potential strategy for alleviating liver I/R injury [16][17][18][19][20]29,37 . In the present study, we found that JNK/p38 phosphorylation was significantly increased in MKP5 KO mice and MKP5 knockdown HepG2 cells, whereas JNK/p38 phosphorylation was decreased in the MKP5 overexpression models. We further examined the changes in the expression of TAK1, a protein upstream of JNK/p38, and MKP5 KO or knockdown significantly increased TAK1 phosphorylation, whereas MKP5 overexpression inhibited the TAK1 phosphorylation. Inhibition of TAK1 activity with a TAK1 inhibitor reversed cell damage caused by MKP5 knockdown. Together, these observations indicate that MKP5 inhibits TAK1 phosphorylation and downstream JNK and p38 activation to alleviate liver I/R injury.
However, this study also has some limitations. In this study, MKP5 gene knockout mice and MKP5 mice in adeno-associated virus overexpressed liver were used. If hepatocyte-specific knockout and overexpression mice were used, the experimental results would be more convincing. In addition, whether MKP5 regulates TAK1 directly or through other means needs to be further investigated.
To conclude, we found that MKP5 ameliorates liver I/R injury through the regulation of inflammation, apoptosis, and oxidative stress. The protective effect of MKP5 is dependent on inhibition of the TAK1/JNK/p38 signaling pathways. Therefore, targeting MKP5 may offer a promising therapeutic approach for liver I/R injury.

Data availability
The data that support the findings of this study are available from the corresponding author on reasonable request.